Assay: Avoiding Interference in Immunoassays

LowCross-Buffer Created to Overcome Bias and Problems Associated with HAMA

Using antibodies for bioanalysis provides high specificity and sensitivity. As a result immunoassays are easy-to-use, robust, and don’t need expensive equipment. Despite all the successful uses, however, there are still problems—namely interference.

Cross-reactivity is defined as specific binding of assay antibodies to molecules other than the analyte. A similar effect, called nonspecific binding, results when the wrong binding molecules are highly concentrated. Human antimouse antibodies (HAMA), which are set to become much more prevalent in the future due to some mouse monoclonal blockbuster drugs, regularly show cross-reactivities to antibodies of other species like rabbit or even goat antibodies leading to effects on nearly any assay reagent.

Interference by heterophilic antibodies like rheumatoid factors show similar effects to HAMA. Such effects are more prevalent in industrialized nations due to the frequency of rheumatism as well as autoimmunity and allergic diseases.

Even assay methods and assay reagents like immunoassay labels can cause interference. In particular, fluorescence dyes used as labels are known to change antibody characteristics. The dyes can lead to aggregation and result in other negative effects on solubility. Such problems can occur even with labeling enzymes.

Endogenous interferers of specimens like albumins, complement factors, lysozymes, and C-reactive protein are well known. Interestingly, the complexity of samples due to viscosity, pH value, salt concentrations, or other characteristics can also result in bias. Matrix effects is the term given to the sum of all interference effects that influence measurement.

Effects

False-positives can be seen easily when the blanks show false-positives as in Figure 1. Unfortunately this is rare. Most false-positives or false-negatives are not easily detectable. More commonly, accuracy and precision are negatively impacted by interference. The FDA’s, Guidance for Industry 2001: Bioanalytical Method Validation, outlines a simple way of showing and detecting those problems.

The reliability of assays for use in preclinical studies is validated according to this FDA guidance. Among other tests, the coefficient of variation (CV) is systematically detected before assays are used for analysis.

Many biopharma firms have problems achieving assay validation that meets the FDA’s acceptance criteria. Among other guidelines, the agency asks for a maximum CV of 15% for validations using real specimens. This has been shown to be hard to achieve for many assays. Even good assay design cannot ensure absolutely reliable measurement. Therefore even experienced assay developers can face interference problems and assays with high CV.

Bad Coefficients of Variation

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Figure 2a

The reasons for interference differ, but most problems have one thing in common—they are derived from low-to-medium affinity binding. Even HAMAs rarely show high affinity, although they are often described as high-affinity binders.

Assay antibodies and analyte should bind to each other with high affinity. Thus a solution to all interference problems would suppress the low-and-medium binding affinities. At the same time such a solution would keep the highest affinities untouched. This binding affinity discriminator would work similar to a wavelength filter in optical physics.

Candor Bioscience’s (www.candor-bioscience.de) LowCross-Buffer® meets these criteria. It is an assay buffer and antibody diluent that enhances the reliability of immunoassays. When antibodies and analytes are incubated in LowCross-Buffer, high-affinity binding is not negatively affected, but low-to-medium affinity binding is depressed. LowCross-Buffer has been shown to be effective in many biomarker assays.

Its ease of use, general applicability in assay formats like ELISA, Western blotting, immunohistochemistry, protein arrays, immuno-PCR, and others, and its applicability with many different matrices like blood, serum, plasma, urine, CSF, tissue specimen, milk, meat, food extracts, and environmental samples make it a useful new tool.

As a result of the specific characteristics of any analyte or matrix, the LowCross-Buffer has to be tested to be effective in any assay with a new validation. Testing is simple because one has only to exchange LowCross-Buffer for the currently used assay buffer or assay diluent. In competitive assays, however, the concentrations of tracer and assay antibodies have to be titered again exactly to the new assay conditions.

LowCross-Buffer is effective. Figure 1 shows avoidance of false-positives in an ELISA. This ELISA against IgG from guinea pigs is used for immunotoxicological studies. In this assay false positives in the specificity control (row A1–A12) as well as at the blank value (H1–H12) spoiled the interpretation and evaluation.

The use of LowCross-Buffer, on the other hand, prevented the false positives and made the detection of concentrations possible in more rows. More often one has assays that don’t show such significant false-positives, but the precision profiles are not in accordance to the acceptance criteria.

Figure 2a shows the precision profile of a biomarker assay. Standard assay buffers could not ensure reliability over the complete concentration range. The interference in this assay is not easy to understand, because the precision profile does not show an expected graph. Such unexpected precision profiles are well known, though. Due to FDA guidance, these assays are validated in a strict manner showing all the erroneous results.

Switching to LowCross-Buffer results in a better precision profile where acceptance criteria are met. Figure 2b shows the CV over the whole measurement range.

HAMA Blocker

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Figure 2b

Commercial HAMA blockers (monoclonal mouse antibodies or fragments thereof in high concentrations) are effective against interference derived from HAMA. They are, however, only effective against this specific kind of interference and they have to be adapted to any new assay.

HAMA blockers are active assay components, which make them extremely critical when lot-to-lot changes occur. This results in a need to revalidate an established assay again with any new batch.

Candor Bioscience has tested LowCross-Buffer with a CE-certified human diagnostic ELISA kit for detection of HAMA reactivity. Complete commercial panels of HAMA-positive sera were tested. Only the results of those sera with detectable HAMA reactivity are shown (Figure 3). The HAMA reactivity of each tested sera was reduced below the level of detection. This analysis showed LowCross-Buffer to be an effective substitute for HAMA blockers.

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Figure 3

Tobias Polifke, Ph.D., is managing director, and Peter Rauch, Ph.D., is head of development at Candor Bioscience. Web: www.candor-bioscience.de.

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